During cell division the mitotic spindle, a multi-component machine, accurately segregates a cell's replicated DNA into two daughter cells. Failure of this process has been linked to numerous developmental defects and oncogenesis. The bipolar organization of the spindle directs movements of sister chromosomes into each daughter cell. The long-term goal of this project is to understand the molecular mechanisms of bipolar spindle assembly. To this end, we have focused our studies on Eg5, an evolutionarily conserved kinesin-related protein required for spindle formation. Loss of Eg5 function during cell division results in monopolar spindles and blocks the cell cycle. Mitosis-specific phosphorylation at a conserved sequence in Eg5 (called the bimC-box) has been proposed to regulate Eg5's spindle targeting. Together with dynein, a minus-end directed microtubule-based motor, and its regulator dynactin, Eg5 organizes spindle microtubules and controls spindle length. We will use Eg5 as a tool to dissect the molecular mechanisms of bipolar spindle formation and force generation in the spindle. Specifically, we will: (1) Examine the influence of microtubule dynamics and organization on the dynamic behavior of Eg5 during spindle assembly, by using fluorescent speckle microscopy and photoactivation of fluorescence. (2) Determine the role of Eg5's motor function in spindle assembly by generating Eg5 mutants with reduced motility and with reduced microtubule affinities, testing these mutants' localization and dynamics in spindles and their ability to rescue spindle formation in Eg5-depleted cell extracts. (3) Analyze the effect of Eg5 phosphorylation on its motor function and structure, by comparing Eg5 phosphorylated at the bimC-box to an Eg5 mutant that cannot be phosphorylated. (4) Determine how dynactin influences Eg5 function in spindles, by examining interactions between dynactin and Eg5 using immunoprecipitation and affinity chromatography, and by comparing the dynamic behavior of dynactin and Eg5 in spindles using fluorescence microscopy. Understanding the mechanism of Eg5 function in spindles should lead to improved inhibitors of Eg5 and could result in better anti-cancer therapeutics.